Alginate is used as a matrix for immunoisolation of cells and tissues in vivo. We have demonstrated previously that commercial alginates contain various fractions of mitogenic impurities and that they can be removed by free flow electrophoresis. The use of purified material is a necessity in order to reveal the parameters that control biocompatibility of the implanted material (such as stability, size, surface charge and curvature, etc.). In this study, we present a protocol for the chemical purification of alginates on a large-scale. Beads made from alginates purified by this multi-step chemical extraction procedure did not induce a significant foreign body reaction when implanted for 3 weeks either intraperitoneally or beneath the kidney capsule of Lewis or non-diabetic BB/Gi rats.

The 5 region of the lac operon of Lactobacillus casei has been investigated. An open reading frame of 293 codons, designated lacT, was identified upstream of lacE. The gene product encoded by lacT is related to the family of transcriptional antiterminator proteins, which includes BglG from Escherichia coli, ArbG from Erwinia chrysanthemi, SacT, SacY, and LicT from Bacillus subtilis, and BglR from Lactococcus lactis. Amino acid sequence identities range from 35 to 24%, while similarities range from 56 to 47%. The transcriptional start site of the lac operon was identified upstream of lacT. The corresponding mRNA would contain in the 5 region a sequence with high similarity to the consensus RNA binding site of transcriptional antiterminators overlapping a sequence capable of folding into a structure that resembles a rho-independent terminator. LacT was shown to be active as an antiterminator in a B. subtilis test system using the sacB target sequence. lacT directly precedes lacEGF, the genes coding for enzyme IICB, phospho-␤-galactosidase, and enzyme IIA, and these genes are followed by a sequence that appears to encode a second rho-independent transcription terminator-like structure. Northern hybridizations with probes against lacT, lacE, and lacF revealed transcripts of similar sizes for the lac mRNAs of several L. casei strains. Since the length of the lac mRNA is just sufficient to contain lacTEGF, we conclude that the lac operon of L. casei does not contain the genes of the accessory tagatose-6-phosphate pathway as occurs in the lac operons of Lactococcus lactis, Streptococcus mutans, or Staphylococcus aureus.Uptake of lactose into bacterial cells and initiation of its metabolism can be mediated by several pathways: ABC protein-dependent systems, lactose-galactose antiporters, lactose-H ϩ symport systems, or the lactose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) (20). While ABC protein-dependent lactose transport has been demonstrated in Agrobacterium radiobacter (59), lactose-galactose antiport has been described for Streptococcus thermophilus (41). The lactose permease-H ϩ symport system, the genetics of which represent the paradigm of bacterial operon organization and regulation (31), is found in the enterobacteria, and its function has been studied in Lactococcus lactis (34). Lactose permease-␤-galactosidase systems have also been identified in other gram-positive species, including lactobacilli (23, 32). The alternative pathway for lactose uptake, however, the lactosespecific PTS (Lac-PTS), has so far been confirmed only in gram-positive bacteria such as Staphylococcus aureus, Streptococcus mutans, dairy lactococci, and Lactobacillus casei strains (2,3,19,20,29,37,40,42,46). In some Lactobacillus species, two pathways, Lactose permease-␤-galactosidase and Lac-PTS, have been found to coexist (23,44).During PTS-mediated transport, lactose is phosphorylated and then hydrolyzed by phospho-␤-galactosidase (P-␤-Gal) to glucose and galactose-6-P. While glucose is channeled into the E...

No abstract

No abstract

Immuno-isolated transplantation offers the attractive prospect of being able to transplant xenogeneic islets without immunosuppression. This study introduces a completely new method of coating single islets using a homogeneous alginate membrane approximately 10 microns thick. During glucose challenge (perifusion and static incubation) encapsulated islets show the same pattern and quantity of insulin release as non-encapsulated controls. This encapsulation method markedly reduces the amount of transplanted material by reducing the size of the capsule. It is suggested that encapsulated islets may be transplanted into sites such as the renal capsule or omentum or even by intraportal injection into the liver.